Construction of improved temperature-sensitive and mobilizable vectors and their use for constructing mutations in the adhesin-encoding acm gene of poorly transformable clinical Enterococcus faecium strains.

Abstract

Inactivation by allelic exchange in clinical isolates of the emerging nosocomial pathogen Enterococcus faecium has been hindered by lack of efficient tools, and, in this study, transformation of clinical isolates was found to be particularly problematic. For this reason, a vector for allelic replacement (pTEX5500ts) was constructed that includes (i) the pWV01-based gram-positive repAts replication region, which is known to confer a high degree of temperature intolerance, (ii) Escherichia coli oriR from pUC18, (iii) two extended multiple-cloning sites located upstream and downstream of one of the marker genes for efficient cloning of flanking regions for double-crossover mutagenesis, (iv) transcriptional terminator sites to terminate undesired readthrough, and (v) a synthetic extended promoter region containing the cat gene for allelic exchange and a high-level gentamicin resistance gene, aph(2'')-Id, to distinguish double-crossover recombination, both of which are functional in gram-positive and gram-negative backgrounds. To demonstrate the functionality of this vector, the vector was used to construct an acm (encoding an adhesin to collagen from E. faecium) deletion mutant of a poorly transformable multidrug-resistant E. faecium endocarditis isolate, TX0082. The acm-deleted strain, TX6051 (TX0082Deltaacm), was shown to lack Acm on its surface, which resulted in the abolishment of the collagen adherence phenotype observed in TX0082. A mobilizable derivative (pTEX5501ts) that contains oriT of Tn916 to facilitate conjugative transfer from the transformable E. faecalis strain JH2Sm::Tn916 to E. faecium was also constructed. Using this vector, the acm gene of a nonelectroporable E. faecium wound isolate was successfully interrupted. Thus, pTEX5500ts and its mobilizable derivative demonstrated their roles as important tools by helping to create the first reported allelic replacement in E. faecium; the constructed this acm deletion mutant will be useful for assessing the role of acm in E. faecium pathogenesis using animal models.

Protocol used for replacing the acm wild-type sequence on the TX0082 chromosome with the cat gene. The gene replacement construct (pTEX6050) carrying in vitro-altered sequences (AcmUp, the region upstream of acm [shown by the cross-hatched box], and AcmDn, part of the acm 3′ region as well as the downstream region [shown by the dotted box]) was transformed into E. coli. At permissive temperature (28°C), pTEX6050 was introduced into TX0082 by electroporation. Colonies were screened for an integration event when the temperature was shifted to 42°C. One of the integrants was grown for eight serial overnight passages at 42°C to completely cure the plasmid. The culture from the eighth passage was serially diluted and plated at 37°C on nonselective media to select for double crossover recombination. Upon replica plating to chloramphenicol plates and gentamicin plates, the colonies that retained the cat gene only were identified. One of these colonies was designated TX6051 (TX0082Δacm). The gray box represents the cat gene coding for chloramphenicol resistance, and the striped box represents the aph(2′′)-Id gene coding for gentamicin resistance.

Confirmation of allelic replacement of the acm gene of TX6051. (A) PFGE analysis of SmaI-digested genomic DNA of TX0082 (lane 1), four double-crossover colonies that retained the chloramphenicol resistance (lanes 2 to 5), a colony resulting from a single-crossover integration event (TX0082AcmUp::pTEX6050) (lane 6), and a molecular weight marker (lane 7). Two fragments marked with straight arrows in lane 6 are due to a SmaI site in the inserted plasmid. The extrachromosomal (nonintegrated) plasmid band is marked with a bent arrow. (B) Illustration of a 4,474-bp Δacm region of E. faecium TX6051 (TX0082Δacm). Sequencing confirmed that 1,663 bp of the acm locus is replaced with a 971-bp cat cassette. The 5′ and 3′ regions that were used for recombination events are shown by cross-striped boxes. (C and D) Southern blot analysis of the digests shown in lanes 1 to 6 of panel A. Panel C was probed with the deleted fragment of acm, and panel D was probed with the cat gene. Hybridization results obtained with pTEX5500ts are identical to those obtained with its cat gene, as shown in panel D.